Bs 7175r

Lung cancer cells or tumor tissues were lysed and sonicated in RIPA buffer supplemented with protease inhibitors (Solarbio, Beijing). Protein extracts were boiled in sample buffer (Solarbio), separated by SDS-PAGE and transferred to nitrocellulose filter membranes (Millipore). After blocking in PBS/Tween-20 containing 5% BSA, the membranes were incubated with the primary antibodies. The following antibodies were used: PCNA (#2586), GAPDH (#5174), p-Erk1/2 (#9101), Erk1/2 (#9102), p-AKT (S473, #9271), AKT (#2920), p-RPS6 (#2215), RPS6 (#2217), p-mTOR (#5536) and mTOR (#2983), purchased from Cell Signaling Technologies; MAL2 from Bioss (bs-7175R). β-Actin (ab179467) was purchased from abcam. Signal visualization was detected with ECL substrate (millipore) and a Biotek imaging system. Immunohistochemistry was performed as previously described [35 (link)].

FTO and MAL2 protein analysis was performed by immunohistochemistry (IHC) staining of bladder cancer tissue microarrays following standard protocol using antibodies (1:100 dilution) against FTO (ab124892, Abcam, Cambridge, MA, USA) and MAL2 (bs‐7175R, BIOSS, Woburn, MA, USA). The H‐score system, based on the percentage of positively stained cells, was used to assess immunoreactivity, and this assessment was performed by two investigators. Based on the immunoreactivity scores, the bladder cancer patients were categorized into the low expression (H‐score < 50%) group and the high expression (H‐score > 50%) group.